Transformed mouse fibroblasts, when grown in chemically defined medium, contained no detectable GM1 and did not accumulate cyclic AP in response to choleragen. Responsiveness to choleragen was observed following the binding of the monosialoganglioside GM1. I125-Choleragen binding was increased dramatically following uptake of GM1, but not GM2 or GM3 by the cells. Although uptake of GM1 was essential for in vivo responsiveness to choleragen, the in vitro activation of adenylate cyclase by choleragen did not require integration of GM1 into the cells. Choleragen binding to cells containing bound exogenous GM1 protected the GM1 from oxidation by galactose oxidase and periodate. Normal human fibroblasts containing endogenous GM1 responded to choleragen, choleragen binding protecting the GM1 from galactose oxidase and periodate. All the endogenous GM1 was not accessible to choleragen or to enzymatic modification by galactose oxidase. Availbility of the GM1 to galactose oxidase, however, could be, increased by disruption of the cells.